# Biochemical Properties and Substrate Specificity of Two Acyl-CoA:Lysophosphatidic Acid Acyltransferases (PtATS2a and PtATS2b) from Diatom Phaeodactylum tricornutum

## TL;DR

This study examines two enzymes from a diatom that help build fats, revealing their optimal conditions and how they choose different fatty acid substrates.

## Contribution

The first biochemical characterization of LPAAT-type enzymes from diatoms, including their substrate specificity and environmental preferences.

## Key findings

- PtATS2a and PtATS2b show highest activity at 23°C and alkaline pH (pH 10 and 11, respectively).
- Both enzymes are inhibited by calcium and high magnesium concentrations.
- PtATS2a prefers 18:4-CoA n-3, while PtATS2b prefers 18:1-CoA as substrates.

## Abstract

Microsomal fractions from yeast Δale1 cells harbouring the empty plasmid pYES2/CT and from yeast cells overexpressing PtATS2a (Phatr3_J11916) or PtATS2b (Phatr3_J43099) were used in the studies. When sn-1-18:1-LPA and [14C]16:0-CoA were used as exogenous substrates, both PtATS2a and PtATS2b showed the highest activity at 23 °C in the range of temperatures tested from 10 to 60 °C. Both enzymes showed the highest activity in alkaline pH. For PtATS2a, it was pH 10 while for PtATS2b, it was pH 11. At pH 6 and pH 12, the activities of both enzymes were very low. The calcium ions at concentrations of 0.05–1 mM drastically decreased the activity of both enzymes. The magnesium ions at a concentration of 0.05 mM had a little effect on the activity of both enzymes, while higher concentrations (0.5 mM and 1 mM) significantly inhibited their activity. To study the substrate specificity, seventeen different acyl-CoAs in combinations with sn-1-[14C]18:1-LPA were used. PtATS2a showed the highest preference for 18:4-CoA n-3 while PtATS2b for 18:1-CoA. The pattern of utilisation of other acyl-CoAs tested also differed between the two enzymes. The presented studies, for the first time, characterised LPAAT type enzymes from diatoms, organisms that naturally produced very-long-chain polyunsaturated fatty acids (VLC-PUFA).